Edward D. T. Atkins

Engineering nanoscale order into a designed protein fiber

We have established a designed system comprising two peptides that coassemble to form long, thickened protein fibers in water. This system can be rationally engineered to alter fiber assembly, stability, and morphology. Here, we show that rational mutations to our original peptide designs lead to structures with a remarkable level of order on the nanoscale that mimics certain natural fibrous assemblies. In the engineered system, the peptides assemble into two-stranded α-helical coiled-coil rods, which pack in axial register in a 3D hexagonal lattice of size 1.824 nm, and with a periodicity of 4.2 nm along the fiber axis. This model is supported by both electron microscopy and x-ray diffraction. Specifically, the fibers display surface striations separated by nanoscale distances that precisely match the 4.2-nm length expected for peptides configured as α-helices as designed. These patterns extend unbroken across the widths (≥50 nm) and lengths (>10 μm) of the fibers. Furthermore, the spacing of the striations can be altered predictably by changing the length of the peptides. These features reflect a high level of internal order within the fibers introduced by the peptide-design process. To our knowledge, this exceptional order, and its persistence along and across the fibers, is unique in a biomimetic system. This work represents a step toward rational bottom-up assembly of nanostructured fibrous biomaterials for potential applications in synthetic biology and nanobiotechnology.

The crystal structure of gellan

Abstract Gellan is a nonsulfated, anionic, extracellular polytetrasaccharide secreted by the bacterium Auromonas elodea . It is potentially useful in the food industry because of its gel-forming properties. The molecular basis of these properties had been investigated by X-ray diffraction analysis of oriented fibers, but an exhaustive study by Upstill et al. in 1986 produced no molecular model with a remotely acceptable fit to the observed X-ray intensities. We describe here a successful re-examination of the crystal structure of gellan; the gellan chains have backbone conformations different from those previously considered. Two left-handed, 3-fold helical chains are organized in parallel fashion in an intertwined duplex in which each chain is translated half a pitch ( p = 5.64 nm) with respect to the other. The duplex is stabilized by interchain hydrogen bonds at each carboxylate group. There are two molecules in each trigonal unit cell ( a = 1.56 nm and c = 2.82 nm).

Structure and Texture of Fibrous Crystals Formed by Alzheimer's Aβ(11–25) Peptide Fragment

Amyloid fibril deposition is central to the pathology of Alzheimers disease. X-ray diffraction from amyloid fibrils formed from full-length Abeta(1-40) and from a shorter fragment, Abeta(11-25), have revealed cross-beta diffraction fingerprints. Magnetic alignment of Abeta(11-25) amyloid fibrils gave a distinctive X-ray diffraction texture, allowing interpretation of the diffraction data and a model of the arrangement of the peptides within the amyloid fiber specimen to be constructed. An intriguing feature of the structure of fibrillar Abeta(11-25) is that the beta sheets, of width 5.2 nm, stack by slipping relative to each other by the length of two amino acid units (0.70 nm) to form beta ribbons 4.42 nm in thickness. Abeta(1-40) amyloid fibrils likely consist of once-folded hairpins, consistent with the size of the fibers obtained using electron microscopy and X-ray diffraction.

Hyaluronates: Relation between Molecular Conformations

The discovery that both potassium and sodium salts of hyaluronic acid can exist in a double-strand helical conformation that will convert to the already known single-strand helical structures illustrates the remarkable conformational versatility of this biopolymer. X-ray diffraction was used to monitor variations in molecular conformation as a function of several independent, controllable variables, such as relative humidity, temperature, and applied tension. A scheme is presented for the interrelation of a range of hyaluronate conformations.

Conformations in polysaccharides and complex carbohydrates

A review is presented focussing attention on the structural molecular biology of polysaccharides and complex carbohydrates, using examples obtained from terraqueous plants, animals, bacteria and insects The type and sequence of the condensation linkages in polysaccharides dominate their conformation, flexibility and interactions The extensive variety of geometries is overlaid by the constituent saccharide units themselves, decoration by side appendages and post-polymerisation chemical and structural modification X-ray diffraction information from oriented samples and computerised modelling has been used to analyse molecular conformation and geometry In general the relationship between glycosidic linkage geometry and conformation for the chemically simpler polysaccharides is understood In the case of more complex carbohydrates, unique solutions using diffraction methods alone are harder to establish In mixed protein carbohydrate systems, such as the glycoprotein antifreezes and protein-polysaccharide fibrous composites in insect cuticle, novel features in structure, morphology and interactions can usefully be explored and examined.

Helical conformations of gellan gum

We have obtained X-ray diffraction patterns of high quality from oriented polycrystalline fibres of the extracellular microbial polysaccharide gellan gum. The diffraction from the lithium salt form indexes on a unit cell with dimensions a=b=1.56±0.03 nm, c (fibre axis)= 2.82±0.05 nm, α=β=90°, γ=120°, with strong meridional reflections observed on the 3rd, 6th and 9th layer lines. The chemical structure consists of a tetrasaccharide repeat: a3)-β-d-Glcp-(1a4)-β-d-GlcpA-(1a4)-β-d-Glcp-(1→4)-α-l-Rhap-(1→. We have examined molecular conformations consistent with this chemical structure, with the measured density of 1.47 g ml−1, the unit cell dimensions and the periodicity and symmetry deduced from the X-ray fibre diffraction patterns. The axial projection of 2.82/3=0.94 nm is unusually short for an unbranched tetrasaccharide repeat (less than half the theoretical maximum), so in addition to very contradicted single helices, a number of intertwined double helix structures capable of generating the appropriate symmetry and layer line spacing were investigated. Computer model building using a linked-atom least-squares (LALS) system yielded four stereochemically acceptable models for a single molecule: a right-handed (31 contracted single helix, right- and left-handed (61 and 65) contracted double helices and a left-handed (32) extended double helix. Examination of the packing of molecules in the unit cell eliminated both 6-fold double helix models. Even after extensive and detailed calculations, the best of each of the surviving models still yield relatively poor agreement between the measured and calculated structure factors. The reliability factors for 3t single helices packed parallel and antiparallel, extended 32 double helices packed parallel and antiparallel were computed to be 0.86, 0.79, 0.71 and 0.65 respectively. This discrepancy between the measured and calculated structure factors can be related to the general distributio of diffracted intensity seen in the X-ray diffraction pattern. Most of the diffracted intensity is concentrated on or close to the equator, and remains so even when the lattice sampling is suppressed by reducing crystallinity whilst maintaining orientation. These observations suggest models with concentrations of electron density on planes closely parallel to the fibre axis, as would be expected from almost fully extended polysaccharide chains. Of all the models considered the extended 32 double helices, packed antiparallel, would be favored on general considerations and best fit with the measured X-ray intensities.

X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium, potassium and calcium cations

Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na+ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds.

Polarized infrared spectra of crystalline glycosaminoglycans.

Polarized infrared spectra have been recorded for oriented, crystalline specimens of hyaluronates, chondroitin 4-sulfate and 6-sulfate, dermatan sulfate, and a cartilage proteoglycan, having different known chain conformations as determined by X-ray diffraction. The dichroism data for the vibrational modes of the amide and carboxyl groups have been interpreted with respect to the particular molecular structures.

Nylon 6 9 can crystallize with hydrogen bonding in two and in three interchain directions

Nylon 6 9 has been shown to have structures with interchain hydrogen bonds in both two and in three directions. Chain-folded lamellar crystals were studied using transmission electron microscopy and sedimented crystal mats and uniaxially oriented fibers studied by X-ray diffraction. The principal room-temperature structure shows the two characteristic (interchain) diffraction signals at spacings of 0.43 and 0.38 nm, typical of α-phase nylons; however, nylon 6 9 is unable to form the α-phase hydrogen-bonded sheets without serious distortion of the all-trans polymeric backbone. Our structure has c and c* noncoincident and two directions of hydrogen bonding. Optimum hydrogen bonding can only occur if consecutive pairs of amide units alternate between two crystallographic planes. The salient features of our model offer a possible universal solution for the crystalline state of all odd–even nylons. The nylon 6 9 room-temperature structure has a C-centered monoclinic unit cell (β = 108°) with the hydrogen bonds along the C-face diagonals; this structure bears a similarity to that recently proposed for nylons 6 5 and X3. On heating nylon 6 9 lamellar crystals and fibers, the two characteristic diffraction signals converge and meet at 0.42 nm at the Brill temperature, TB · TB for nylon 6 9 lamellar crystals is slightly below the melting point (Tm), whereas TB for nylon 6 9 fibers is ≅ 100°C below Tm. Above TB, nylon 6 9 has a hexagonal unit cell; the alkane segments exist in a mobile phase and equivalent hydrogen bonds populate the three principal (hexagonal) directions. A structure with perturbed hexagonal symmetry, which bears a resemblance to the reported γ-phase for nylons, can be obtained by quenching from the crystalline growth phase (above TB) to room temperature. We propose that this structure is a “quenched-in” perturbed form of the nylon 6 9 high-temperature hexagonal phase and has interchain hydrogen bonds in all three principal crystallographic directions. In this respect it differs importantly from the γ-phase models.

Biomolecular structures of naturally occurring carbohydrate polymers

Abstract The bonding mechanism in proteins and polysaccharides is compared and contrasted. Some of the structures for polyglucose are shown to mimic the classic β-sheet, α-helix and collagen helices found in proteins. The effects of glycosidic linkage geometry on polysaccharide shape and structure is discussed together with the effect of changes in the geometry of the monomer units. Multistranded ropes and the effect of non-carbohydrate groups are mentioned briefly.